Nitric oxide is synthesized enzymatically from L-arginine by almost all tissues of the body, including brain, peripheral nervous system, smooth muscle, kidney, vascular, lung, uterus, etc. Nitric oxide is involved in many vascular functions including vasorelaxation and blood clotting. (Moncada et al., 1993). Heme proteins such as guanylyl cyclase serve as primary targets for nitric oxide (Bredt et al., 1994).
There is a substantial body of evidence from animal and human studies that a deficiency in nitric oxide contributes to the pathogenesis of a number of diseases, including hypertension, artherosclerosis, and diabetes.
Depending upon the rate, timing, and spatial distribution of nitric oxide production, as well as the chemical microenvironment (e.g., presence of ROS, redox status of the cell), nitric oxide acts either as a direct signaling messenger or as an indirect toxic effector via the formation of various reactive nitrogen species. Indeed, both functions may be occurring at the same time, i.e., spatially targeted production of nitric oxide may simultaneously enhance the antimicrobial function of the neutrophil respiratory burst while protecting host cells from oxidant injury and, perhaps, preventing the uncontrolled explosion of the inflammatory response (e.g., down-regulation of nuclear factor κ-B activation). At other times, the cytotoxic potential of reactive nitrogen species may turn against the host, as has been documented in some murine models of infection, such as Influenza A pneumonitis or intestinal and liver injury caused by T. gondii. (Feihl et al., 2001).
Nitric oxide was originally described as the principal endothelium-derived relaxing factor, but it is now known to subserve a variety of functions throughout the body, both physiological and pathophysiological. Among its diverse functions, nitric oxide has been implicated in neurotransmission, immune regulation, vascular smooth muscle relaxation, and inhibition of platelet aggregation.
Nitric oxide has been found to activate soluble guanylate cyclase. Guanylate cyclase exists in two forms in the cell: a soluble isoform within the cytosol and a particulate isoform which is membrane-associated. Purified soluble guanylate cyclase can be activated in vitro by nitric oxide-donating compounds. Once the enzyme has been activated, there is accumulation of cGMP which, in vascular smooth muscle cells, is accompanied by relaxation and hence vasodilation.
In other cell types, the accumulation of cGMP is accompanied by different physiological effects. cGMP can be formed by soluble guanylate cyclase in platelets, following stimulation by nitric oxide derived either from the vascular endothelium or from endothelial-type NOS within the platelets themselves, leading to inhibition of platelet aggregation. In the central nervous system, nitric oxide can be formed in the postsynaptic nerve cell following activation of N-methyl-D-aspartate (NMDA) receptors by glutamate. This nitric oxide diffuses out and acts upon guanylate cyclase in one or more neighboring neurons, including the presynaptic nerve cells, thus acting as part of a feedback loop. In one model of brain activity, such a retrograde messenger is involved in the building up of long-term memory. In addition, nitric oxide in the brain can act upon nerve cells other than the presynaptic nerve cell, causing a variety of effects: neuroprotection, long-term potentiation (an activity-dependent increase in synaptic strength), and long-term depression (a long-lasting depression of parallel fiber synapses following repeated excitation by climbing fibers of Purkinjie cells). It has also been suggested that overproduction of nitric oxide may cause neurotoxicity and certain degenerative conditions, such as Alzheimer-type dementia, either because of its radical nature or because it can generate peroxynitrite.
Nitric oxide is generated in the course of inflammatory and immune reaction, both by macrophages and by neutrophils. The role of these cells is quite extensive, and includes phagocytic and non-phagocytic destruction of foreign or damaged cells. These processes involve the production of large quantities of nitric oxide, which is cytotoxic.
It is possible that either an overabundance or a deficiency of nitric oxide is involved in many other pathological problems in females, such as preeclampsia, preterm labor, climacterium, pregnancy-induced diabetes, and postpartum hemorrhage. Nitric oxide overabundance or deficiency may also be associated with coronary artery disease, cancer, and behavioral and digestive problems.
Premenopausal women have a lower incidence of cardiovascular disease than men. After menopause, the incidence of cardiovascular disease increases progressively, as the risk of coronary heart disease rapidly increases after cessation of ovarian function. These changes are thought to be hormonally mediated and related to the decrease in production of both estrogen and progesterone. Since nitric oxide is very important in control of vascular function, a decrease in nitric oxide production or action is related to pathophysiological changes in blood vessels, i.e., cardiovascular diseases associated with hypertension and artherosclerosis. The steroid hormones regulate nitric oxide synthesis. Thus, it is believed that nitric oxide may mediate all, or at least some, of the actions of the steroid hormones to prevent cardiovascular disease in premenopausal women. It may thus be possible to administer a nitric oxide donor to prevent cardiovascular disease as part of or instead of hormone replacement therapy, thus minimizing or eliminating any undesirable side effects of hormone replacement therapy.
Nitric oxide is also very much involved in the control of blood clotting. Nitric oxide and its donors are potent inhibitors of coagulation (e.g., reviewed in Richardson G. and Benjamin N. (2002)).
In addition, nitric oxide has been implicated in bone remodeling. Bone remodeling disorders such as osteoporosis and osteoarthritis are frequently associated with perturbations in the interaction between local and systemic bone-remodeling regulatory pathways. Postmenopausal bone loss associated with diminished steroid hormones is correlated with increased levels of cytokines. In addition, both estrogen and progestins are effective in preventing postmenopausal bone loss (Garfield, et al., 1999).
Bone-degrading osteoclasts arise from cells within the monocyte macrophage lineage. Excessive osteoclast activity leads to high levels of bone destructions and osteoporosis. Although these cells have the unique ability to resorb bone, they share various characteristics with macrophages (Garfield, et al., 1999).
Macrophages release nitric oxide in response to inflammatory cytokines and agents. It has been suggested that osteoclasts, like macrophages, synthesize nitric oxide. In models of osteoporosis, nitric oxide inhibition potentiated the loss of bone mineral density by suppressing osteoclast activity and bone resorption. There have also been studies which demonstrated that nitric oxide is produced by chondrocytes. (Garfield, et al., 1999).
Since inhibition of osteoclastic activity is a major aim in treating and preventing osteoporosis, Paget bone disease and rheumatoid arthritis, nitric oxide donors may be useful in treating and preventing these conditions.
Presently, there are only three nitric oxide donor compounds that are used clinically: nitroglycerin, amyl nitrite, and sodium nitroprusside. Nitroglycerin is available in tablets or sprays for sublingual use, intravenously, or in patch form. Amyl nitrite is formulated as an inhalant and is usually used in breakable capsules. Sodium nitroprusside is limited to intravenous infusion. Nitric oxide donors are currently used for treating angina pectoris due to coronary artery disease (nitroglycerin or amyl nitrite) and control of blood pressure associated with myocardial infarction or surgical procedures (nitroglycerin or sodium nitroprusside).
Unfortunately, conventionally available nitric oxide donor compounds have a short duration of action, a short half-life, a lack of tissue specificity, development of tolerance, and accumulation of toxic substances (cyanide for sodium nitroprusside).
Nitric oxide is a gas with low solubility in water and aqueous solutions. Although nitric oxide is a free radical that is highly unstable in vivo, it does not interact directly with most biological substrates and commonly used organic solvents.
An important physiologically significant component of nitric oxide biochemistry involves formation of thionitrite esters with free thiols (S-nitrosothiols: RS-No). Low molecular weight S-nitrosothiols, e.g., S-nitrosoglutathione (GS-NO), S-NO-cysteine (S-NO-Cys) and nitroso derivatives of proteins such as albumin and hemoglobin (Hb) exert nitric oxide-like activity in vivo. They cause arterial and venous smooth muscle relaxation, inhibit platelet aggregation, and activate guanylate cyclase (Stamler et al., 1992; Ignarro et al., 1981; Keaney et al., 1993; Stamler, 1992; Scharfstein et al., 1994; Jia et al., 1996).
Vasoactive S-nitrosothiols are known to be generated in vivo (Stamler et al., 1992; Ignarro et al., 1981; Keaney et al., 1993; Stamler, 1992; Scharfstein et al., 1994; Jia et al., 1996; Clancy et al., 1994; Gaston et al., 1998). The originally reported amount of total S-nitrosothiols in human plasma was 7 μM (Stamler et al., 1992). Many subsequent measurements (Rafikova et al., 2002) detected from 40 nM to 1 μM of plasma S-nitrosothiols in humans and rodents under noninflammatory conditions (Hogg et al., 2002; Marzinzig, 1997; Tsikas et al., 1999). Since S-nitrosothiol compounds are relatively stable and can release nitric oxide when required via reactions with transition metal ions or other reducing agents (Singh et al., 1996; Kashiba-Iwatsuki et al., 1997; Aleryani et al., 1998; Nikitovic et al., 1996), they are envisioned as a buffering system that controls intra- and extra-cellular activities of NO and magnify the range of its action. Once formed, S-nitrosothiols can directly transfer the nitrosyl cation (NO+) to another thiol via the so-called transnitrosation reaction, which ensures the dynamic state of S-nitrosothiols in vivo (Ignarro et al., 1981; Jourd'heuil et al., 2000; Tsikas et al., 2001).
The properties of S-nitrosothiols (RSNOs) are similar to those of nitric oxide. They are involved in smooth muscle cell relaxation, platelet deactivation, immunosuppression, neurotransmission, and host defense. Although they have not yet been used therapeutically, there are data to suggest that S-nitrosothiols have a possible place in the management of a variety of diseases.
At present, the only commercially available S-nitrosothiols are S-nitroso-N-acetylpenicillamine (SNAP), N-acetyl-S-nitrosopenicillaminyl-S-nitrosopenicillamine, and GSNA. None of these compounds has as yet been used therapeutically in animals or humans. One of the main problems with using S-nitrosothiols is their unpredictable rate of decomposition in physiological vehicles, which can occur in the presence of copper and other divalent metals, by enzymatic degradation and as a result of transnitrosation.
Other S-nitrosothiols proposed as drugs are SNO-albumin, which acts as a reservoir of nitric oxide. It is formed when transnitrosation occurs between albumin and low-molecular-mass RSNOs, such as GSNO or SNO-cysteine. However, the cysteine residues in native albumin are few and are hidden inside the molecule. Different forms of poly-SNO-albumin have been prepared by covalent modification of albumin prior to nitrosylation.
Poly-SNO-albumin has been shown to inhibit human vascular smooth muscle cell proliferation in culture to a greater extent than both SNO-albumin and the NONOates.
Captopril, an agiotensin-converting enzyme (ACE) inhibitor, is a reduced thiol and can be nitrosated. It still acts as an ACE inhibitor in this form, but it also functions as a nitric oxide donor, being more effective than captopril itself in decreasing acute and chronic elevation of blood pressure in rats. SNO-captopril can also take part in transnitrosation reactions, transferring its nitroso moiety to haem proteins.
Tissue plasminiogen activator (tPA) is another drug used in routine clinical practice that can undergo nitrosation. This has no effect on its catalytic ability, fibrin stimulation, binding to fibrinogen, or interaction with plasminogen activator inhibitor-1. It does, however, have vasodilatory and anti-platelet effects by enhancing cGMP production. SNO-tPA was compared with tPA in cats in which ischemia/reperfusion injuries were induced. Treatment with SNO-tPA lowered the amount of myocardial necrosis and improved preservation of endothelial function, as assessed by relaxation in response to acetylcholine.
Von Willebrand factor is involved in platelet adhesion. A fragment of von Willebrand factor has been found to have a point mutation, where arginine is replaced by cysteine, and thus can be nitrosated. The fragment itself has anti-platelet properties, but nitrosation improves its ability to inhibit platelet aggregation and adhesion, both in vitro and in an ex vivo rabbit model. All of these effects were mediated both by enhancement of cGMP levels and by decreased binding to glycoprotein (Gp)Ib receptors.
Platelets play a significant role in both the development and the clinical presentation of vascular disease. Currently available agents that reduce platelet activation and adhesion, including aspirin, copidogrel, and abciximab, result in improved outcomes in patients with acute coronary syndromes. However, absolute levels of morbidity and mortality remain high. All RSNOs have been shown to influence platelet function. cGMP is involved in the anti-platelet effects of RSNOs, but, for GSNO, at least, other mediators also appear to be involved. Unlike organic nitrates, which can also lower platelet activity, but at high doses, RSNOs can achieve this in healthy human volunteers in doses that do not affect vascular tone.
Platelet activation, as measured by levels of expression of the adhesion molecule P-selectin and the GpIIb/IIIa receptor, is increased in acute coronary syndromes. This phenomenon is seen even in the presence of aspirin. In a small clinical trial, it was found that GSNO significantly lowered levels of platelet activity. Glyceryl trinitrate also achieved this effect but, because it also induced hypotension, it was less well tolerated.
Interventional treatment of vascular disease using balloon angioplasty and stenting or coronary artery bypass grafting results in platelet activation. This is thought to play a role in the re-stenitic process observed following percutaneous intervention and in graft failure after surgery. SNO-albumin delivered locally to an area of balloon-injured rabbit femoral artery reduced platelet deposition and the subsequent development of neointimal hyperplasia. Local delivery of SNO-albumin using stent-based rather than catheter-based therapy was also found to reduce platelet adhesion following deployment into pig carotid arteries. In a small clinical study, intracoronary infusion of GSNO prior to percutaneous transluminal coronary angioplasty prevented the increases in platelet P-selectin and GpIIb/IIIa expression usually seen within five minutes of the procedure, without altering blood pressure. Coronary artery bypass grafting is also associated with platelet activation and consumption, which can lead to post-operative bleeding. GSNO decreased the uptake of platelets by both arteries and veins in vitro and in patients undergoing coronary artery bypass grafting.
One of the main complications of carotid endarterectonmy is cerebral infarction, often caused by platelet emboli. The procedure itself results in the removal of the endothelium, leaving a potent thrombogenic surface on which platelet adhesion and aggregation occurs. Asymptomatic microemboli can be detected by Doppler ultrasonography, and their frequency correlates with the risk of early stroke. In small studies, intravenous GSNO given either peri-operatively or post-operatively reduced the frequency of microemboli compared with placebo.
Like nitric oxide, RSNOs relax vascular smooth muscle cells. Another therapeutic use for RSNOs is in managing subnarachnoid hemorrhage, a condition associated with cerebral vasospasm. This vascular response was significantly reduced by an infusion of SNAP in a rat model of subarachnoid hemorrhage.
Coronary artery bypass grafting involves the use of both saphenous vein an internal mammary arteries as bypass conduits. Handling of these tissues can include vasospasm, potentially leading to early graft occlusion. Rings of conduit taken from patients undergoing coronary artery bypass grafting were exposed to GSNO and RIG200, another RSNO, and their vascular responses were compared with that seen after exposure to GTN. The RSNOs were found to significantly reduce the degree of vasospasm in these tissue sections compared with GTN.
Reperfusion of ischemic tissue leads to inflammatory responses and endothelial cell dysfunction. RSNOs have been shown to improve end-organ recovery in models of ischemia/reperfusion injury in the heart and the liver.
Thus, RSNOs have many potential roles in the treatment of vascular diseases, limiting the complications of platelet activation, of vasospasm, and of ischemia/reperfusion. These agents may be more effective than currently available nitric oxide donors, such as organic nitrates, which have generally shown little benefit apart from symptomatic relief.
Normal levels of RSNOs detected in bronchoalveolar lavage fluid are approximately 25 μM. RSNOs relax bronchial smooth muscle, inhibiting the broncho-constrictor effects of methacholine on segments of human airway. As with their anti-platelet actions, cGMP appears to mediate only part of this bronchodilatory effect of RSNOs.
Airway RSNO levels are altered in disease states. In patients with pneumonia, the mean concentration was 0.4 μM, higher than that seen in healthy controls. In patients with asthma, nitric oxide levels in exhaled breath are higher than normal, but airway RSNO levels are much reduced as compared with healthy controls.
Like nitric oxide, RSNOs appear to play a role in host defense, affecting both bacteria and viruses. Macrophages produce nitric oxide from L-arginine to exert a cytostatic effect in limiting the complications of platelet activation, of vasospasm, and of ischemia/reperfusion. These agents may be more effective than currently available nitric oxide donors, such as organic nitrates, which have generally shown little benefit apart from symptomatic relief.
Normal levels of RSNOs detected in bronchoalveolar lavage fluid are approximately 25 μM. RSNOs relax bronchial smooth muscle, inhibiting the broncho-constrictor effects of methacholine on segments of human airway. As with their anti-platelet actions, cGMP appears to mediate only part of this bronchodilatory effect of RSNOs.
Airway RSNO levels are altered in disease states. In patients with pneumonia, the mean concentration was 0.4 μM, which was higher than that seen in healthy controls. In patients with asthma, nitric oxide levels in exhaled breath are higher than normal, but airway RSNO levels are much reduced as compared with healthy controls.
Like nitric oxide, RSNOs appear to play a role in host defense, affecting both bacteria and viruses. Macrophages produce nitric oxide from L-arginine to exert a cytostatic effect on Trypanosoma musculi. This effect is not seen in the absence of albumin, or in the presence of antibodies to SNO-cysteine, suggesting that the nitric oxide reacts with albumin to form SNO-albumin and then a trans-nitrosation reaction with cysteine occurs to form SNO-cysteine, the active moiety. RSNOs are toxic to a mutant form of Salmonella typhimurium in which intracellular homocysteine levels are depleted.
Both nitric oxide and SNAP inhibit HIV-1 protease, an enzyme involved in the replication of this virus. When nitric oxide reacts with the enzyme, it forms nitrosothiols with the two cysteine residues in the protein molecule. This effect on HIV-1 is cGMP-independent and additive with that of zidovudine.
GSNO, SNAP, and SNO-captopril inactivate a protease enzyme in the human rhinovirus, which causes the common cold. This effect probably involves a transnitrosation reaction.
Accordingly, RSNOs can be used to treat conditions ranging from the common cold to AIDS.
RSNOs are inhibitors of gastrointestinal smooth muscle function. Their formation in the gut is thought to occur directly as a result of nitrosation of SH groups of low-molecular-mass thiols. Inhibition of motor activity in the duodenum and sphincter of Oddi is a goal during endoscopic retrograde cholangio-pancreatography. Systemically administered GTN has been shown previously to lower basal tone in the sphincter of Oddi during such procedures, although its use is limited by the development of systemic hypotension. Topical application of S-nitroso-N-acetylcysteine to the ampulla and peri-ampullary duodenal mucosa of humans undergoing endoscopic retrograde cholangio-pancreatoraphy caused a reduction in the frequency of sphincter contractions and in duodenal motility, without a fall in blood pressure, thus facilitating cannulation.
GNSO has been shown to inhibit DNA synthesis and to increase cGMP production by activated T lymphocytes, thus suggesting a role in the prevention of T-cell mediated inflammation. GNSO also has a cytotoxic effect on leukemia cells. Irradiation of GNSO with visible light (340 or 545 nm) resulted in enhancement of this effect, and oxyhemoglobin, a nitric oxide scavenger, decreased it.
Because of their wide range of effects, RSNOs have great potential as therapeutic agents. However, interactions with copper, thiols, ascorbic acid, and other reducing agents influences their stability and ability to act as useful therapeutic agents.
Nitric oxide is unable to react with nucleophiles such as SH groups under oxygen-free conditions, implying that metabolites of NO oxidation, such as N2O3, are the actual nitrosating agents (Wink et al., 1994; Lewis et al., 1994; Kharitonov et al., 1995; Williams, 1997). However, considering the low concentration and short life span of NO in vivo (t1/2˜0.1 s) (Williams, 2001), the third order reaction of NO with oxygen (Moncada et al., 1993), (k=6×106 M−2 sec−1) (Wink et al., 1994; Lewis et al., 1994) seems to be too slow to account for any detectable amount of circulating S-nitrosothiols. The high instability of N2O3 in aqueous solution (Stamler et al., 1992) further supports this notion.
Recently, these apparent theoretical constraints have been resolved by the demonstration of remarkable properties of NO in multi-phase systems (Rafikova et al., 2002; Nedospasov et al., 2000). NO and O2 are both hydrophobic molecules that are more soluble in lipophilic solvents than in water, with a partition coefficient Q in each case >>1. Areas of high hydrophobicity act as a sponge to increase the local concentration of NO and O2 by sequestering them from the surrounding aqueous phase. Under aerobic conditions, high local concentrations of NO and O2 in the hydrophobic phase, e.g., within lipid membranes, can significantly accelerate NO oxidation and N2O3 formation (Nedospasov et al., 2000; Liu et al., 1998; Goss et al., 1999). This process, known as micellar catalysis, must play a crucial role in NO biochemistry (see FIG. 1).
Body fluids and tissues represent a complex multiphase system where distribution of free NO and O2 and the rate of formation of nitrosating species (N2O3) should depend on the size and geometry of the hydrophobic phases. Other than lipid membranes, there are many substances that form hydrophobic micelles in vivo, e.g., cholesterol, fatty acids, and hydrophobic cores of protein molecules, with a different Q in each case. Recently, it has been demonstrated that micellar catalysis of NO oxidation is mediated by serum albumin, which has a QNO˜120 (Nedospasov et al., 2000). Albumin is the most abundant transport and depot protein in the circulation. Since the concentration of albumin in plasma is large (˜800 μM), its hydrophobic core serves as a major absorber of free NO and a catalyst of N2O3 (Nedospasov et al., 2000). This view is further supported by data which demonstrate that albumin is an efficient catalyst of nitrosation of its own Cys34 and Trp230 (Nedospasov et al., 2000) as well as circulating low molecular weight RSH (Rafikova et al., 2002).
Since S-nitrosothiols compounds are potent vasodilators and antiplatelet agents, they are considered to be promising therapeutics for a variety of acute and chronic conditions (Hogg, 2000; Al-Sa'doni et al., 2000; Richardson et al., 2002). Understanding the principles of S-nitrosothiol formation in vivo makes it possible to design new approaches to regulate blood flow and clotting and other processes dependent upon nitric oxide. One such an approach is the use of a synthetic hydrophobic phase, such as perfluorocarbons, for the catalysis of NO oxidation and nitrosothiols formation.
Perfluorocarbons are chemically inert, synthetic hydrophobic molecules that possess a unique capability for dissolving a variety of gases, including O2, CO2, and NO. Most therapeutic uses of fluorocarbons are related to the remarkable oxygen-carrying capacity of these compounds. One commercial biomedical perfluorocarbon emulsion, Fluosol (Green Cross Corp., Osaka, Japan) is presently used as a gas carrier to oxygenate the myocardium during percutaneous transluminal coronary angioplasty. Perfluorocarbon emulsions have also been used in diagnostic applications such as imaging.